补肾活血汤对骨髓间充质干细胞迁移及趋化因子受体4,趋化因子13表达的影响
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摘要
目的:探讨补肾活血汤及其拆方对骨髓间充质干细胞(BMSCs)迁移及趋化因子(CXCL)13及趋化因子受体(CXCR)4表达的影响。方法:以大鼠BMSCs为研究对象,制备含药血清,采用全骨髓贴壁法培养BMSCs。通过Transwell实验观察补肾活血汤(全方组)及其拆方(补肾组、活血组)含药血清干预BMSCs体外迁移的能力,采用PCR法检测CXCL13及CXCR4RNA表达水平,采用WesternBlot法检测CXCR4蛋白表达水平。结果:通过培养获得高纯度的BMSCs.Transwell实验显示:全方含药血清干预的大鼠BMSCs,其迁移能力显著增强,与其它4组对比差异均有统计学意义(P<0.05)。PCR结果显示:补肾活血汤全方含药血清能显著上调大鼠BMSCs的CXCL13及CXCR4RNA表达水平,与其它4组比较,差异均有统计学意义(P<0.05)。Western Blot结果显示:补肾活血汤全方含药血清能显著上调大鼠BMSCs的CXCR4蛋白表达水平,与其它4组比较,差异均有统计学意义(P<0.05)。结论:全方组含药血清促BMSCs体外迁移的影响较显著,结合其影响CXCL13和CXCR4的表达水平,较其单纯的活血或补肾组效果更显著,其促进BMSCs体外迁移机制可能与同时上调CXCL13及CXCR4表达,从而激活CXCL13/CXCR5信号轴及SDF-1/CXCR4信号轴有关。合理配伍促进了补肾活血汤促BMSCs体外迁移的能力。
Objective:To explore the effect of Bushen Huoxue Decoction(BHD)and it's separated prescription medicated on the migration of bone marrow mesenchymal stem cells(BMSCs),and expression of chemokine receptor CXCL13 and chemokine receptor CXCR4.Methods:BMSCs were used as the cell model,and were cultivated by bone marrow adherent method.Transwell assay was used to observe the effects of serum containing different dosages of BHD and it's separated prescription medicated on cell migration of BMSCs.The expression levels of chemokine receptor CXCL13 and chemokine receptor CXCR4 RNA were detected by PCR.The expression level of chemokine receptor CXCR4 protein was detected by Western Blot.Results:High purity BMSCs was obtained by culture.Transwell results showed that the whole prescription group containing serum in BHD could significantly enhance the migration ability of BMSCs in rats,and the difference was statistically significant compared with the other four groups.PCR results showed that the whole prescription group containing serum in BHD could significantly increase the level of CXCL13 and CXCR4 expression of BMSCs in rats,and the difference was statistically significant compared with the other four groups(P<0.05).Western blot results showed that the whole prescription group containing serum in BHD could significantly increase the level of CXCR4 protein expression of BMSCs in rats,and the difference was statistically significant compared with the other four group(P <0.05).Conclusion:The serum containing BHD could promote BMSCs migration,and regulate the expression level of CXCL13 and CXCR4,was statistically significant compared with the separated prescription medicated groups,and its mechanism may be related to the simultaneous increase of CXCR4 expression and CXCL13 expression,thereby activating SDF-1/CXCR4 axis and CXCL13/CXCR5 axis.Rational compatibility promotes the ability of Bushen Huoxue Decoction to promote BMSCs migration
Objective:To explore the effect of Bushen Huoxue Decoction(BHD)and it's separated prescription medicated on the migration of bone marrow mesenchymal stem cells(BMSCs),and expression of chemokine receptor CXCL13 and chemokine receptor CXCR4.Methods:BMSCs were used as the cell model,and were cultivated by bone marrow adherent method.Transwell assay was used to observe the effects of serum containing different dosages of BHD and it's separated prescription medicated on cell migration of BMSCs.The expression levels of chemokine receptor CXCL13 and chemokine receptor CXCR4 RNA were detected by PCR.The expression level of chemokine receptor CXCR4 protein was detected by Western Blot.Results:High purity BMSCs was obtained by culture.Transwell results showed that the whole prescription group containing serum in BHD could significantly enhance the migration ability of BMSCs in rats,and the difference was statistically significant compared with the other four groups.PCR results showed that the whole prescription group containing serum in BHD could significantly increase the level of CXCL13 and CXCR4 expression of BMSCs in rats,and the difference was statistically significant compared with the other four groups(P<0.05).Western blot results showed that the whole prescription group containing serum in BHD could significantly increase the level of CXCR4 protein expression of BMSCs in rats,and the difference was statistically significant compared with the other four group(P <0.05).Conclusion:The serum containing BHD could promote BMSCs migration,and regulate the expression level of CXCL13 and CXCR4,was statistically significant compared with the separated prescription medicated groups,and its mechanism may be related to the simultaneous increase of CXCR4 expression and CXCL13 expression,thereby activating SDF-1/CXCR4 axis and CXCL13/CXCR5 axis.Rational compatibility promotes the ability of Bushen Huoxue Decoction to promote BMSCs migration
引文
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[8]刘亚梅,王斌,罗毅文,等.补肾活血法调控大鼠骨髓间充质干细胞增殖活性的实验研究[J].中国中医骨伤科杂志,2015,23(3):1-4.
[9]郭启发,李光,任荣,等.骨髓间充质干细胞对创伤性骨折愈合的促进作用[J].中国组织工程研究,2016,45(3):130-134.
[10]LUTTICHAU IV,NOTOHAMIPRODJO M,WECH-SELBERGER A,et al.Human adult CD34-progenitor cells functionally express the chemokine receptors CCR1,CCR4,CCR7,CXCR5,and CCR10but not CX-CR4[J].Stem Cells Dev,2005,14(3):329-336.
[11]易诚青,姚爱华,马春辉,等.装载趋化因子CXCL13的中空羟基磷灰石微球对间充质干细胞的趋化效应研究[J].上海交通大学学报:医学版,2012,32(12):1532-1534.
[12]付纳新,马超,孙劲,等.臭氧联合关节镜对膝关节骨关节炎滑膜IL1RI、CXCL13与IL24的影响[J].实用医学杂志,2017,33(18):3055-3058.
[13]王羿,党兴.补肾活血法对SD大鼠骨折模型愈合影响的实验研究[J].时珍国医国药,2012,23(12):3150-3151.
[14]陆吴超,钱伟宏,姚志宏,等.补肾、活血中药及补肾活血复方对骨髓间充质干细胞增殖、定向迁移及成骨分化的影响及作用机制的研究进展[J].中医正骨,2018,30(7):36-37.
[15]黄永铨,罗毅文,王斌,等.补肾活血汤治疗老年桡骨远端骨折的临床疗效观察[J].中国中医骨伤科杂志,2015,23(3):5-8.
[16]程英雄,罗毅文,王斌,等.补肾活血汤治疗老年肱骨近端骨折围手术期的应用研究[J].中国中医骨伤科杂志,2016,24(10):16-19.
[17]GONG J,MENG HB,HUA J,et al.The SDF-1/CXCR4axis regulates migration of transplanted bone marrow mesenchymal stem cells towards the pancteas in rats with acute pancreatitis[J].Mol Med Rep,2014,9(5):1575-1582.
[18]GHADGE S K,MUHLSTEDT S,OZCELIK C,et al.SDF-1alpha as a therapeutic stem cell honing factor in myocardial infarction[J].Pharmacol Ther,2011,129(1):97-108.
[19]LISIGOLI G,TONEGUZZI S,GRASSI F,et al.Different chemokine are expressed in human arthritis bone biopsies:IFN-γand IL-6 differently modulate IL-8,MCP-1 and RANTES production by arghritis osteoblasts[J].Cytokine,2002,20(5):231-238.
[20]SIMTH H,WHITTALL C,WEKSLER B,et al.Chemokines stimulate bidirectional of human mesenchymal stem cells across bone marrow endothelial cells[J].Stem Cells Dev,2012,21(3):476-486.
[21]易诚青,马春辉,张国桥,等.CXCL13和CXCR5在骨缺损成骨微环境中的表达特征[J].上海交通大学学报:医学版,2012,32(12):1536-1539.
[22]李新建,齐振熙,李志敏,等.补肾活血汤含药血清骨髓间充质干细胞成骨-成脂分化的影响[J].中华中医药杂志,2016,24(8):3274-3277.
[2]杨丽,朱晓峰,张荣华.补肾活血复方含药血清对MSCs增殖和骨向分化影响的时效及量效研究[J].中药材,2012,35(2):259-262.
[3]GOLPANIAN S,EL-KHORAZATY J,MENDIZABALA,et al.Effect of aging on human mesenchymal stem cell therapy in ischemic cardiomyopathy patients[J].J Am Coll Cardiol,2015,65(2):125-132.
[4]鲍青青,钱伟宏,姚志宏.补肾活血汤治疗老年肱骨近端骨折的临床观察[J].浙江临床医学,2018,20(8):1430-1431.
[5]ITO H.Chemokines in mesenchymal stem cell therapy for bone repair:a novel concept of recruiting mesenchymal stem cells and the possible cell source[J].Mod Rheumatol,2011,21(2):113-121.
[6]熊云谱,王斌,刘亚梅,等.补肾活血汤含药血清对骨髓间充质干细胞迁移及CXCR4表达的影响[J].广州中医药大学学报,2017,34(1):76-79.
[7]罗毅文,王斌,吴志方,等.补肾活血汤提取物促进骨髓间充质干细胞体外迁移及CXCR4表达的研究[J].中药新药与临床药理,2016,27(3):357-359.
[8]刘亚梅,王斌,罗毅文,等.补肾活血法调控大鼠骨髓间充质干细胞增殖活性的实验研究[J].中国中医骨伤科杂志,2015,23(3):1-4.
[9]郭启发,李光,任荣,等.骨髓间充质干细胞对创伤性骨折愈合的促进作用[J].中国组织工程研究,2016,45(3):130-134.
[10]LUTTICHAU IV,NOTOHAMIPRODJO M,WECH-SELBERGER A,et al.Human adult CD34-progenitor cells functionally express the chemokine receptors CCR1,CCR4,CCR7,CXCR5,and CCR10but not CX-CR4[J].Stem Cells Dev,2005,14(3):329-336.
[11]易诚青,姚爱华,马春辉,等.装载趋化因子CXCL13的中空羟基磷灰石微球对间充质干细胞的趋化效应研究[J].上海交通大学学报:医学版,2012,32(12):1532-1534.
[12]付纳新,马超,孙劲,等.臭氧联合关节镜对膝关节骨关节炎滑膜IL1RI、CXCL13与IL24的影响[J].实用医学杂志,2017,33(18):3055-3058.
[13]王羿,党兴.补肾活血法对SD大鼠骨折模型愈合影响的实验研究[J].时珍国医国药,2012,23(12):3150-3151.
[14]陆吴超,钱伟宏,姚志宏,等.补肾、活血中药及补肾活血复方对骨髓间充质干细胞增殖、定向迁移及成骨分化的影响及作用机制的研究进展[J].中医正骨,2018,30(7):36-37.
[15]黄永铨,罗毅文,王斌,等.补肾活血汤治疗老年桡骨远端骨折的临床疗效观察[J].中国中医骨伤科杂志,2015,23(3):5-8.
[16]程英雄,罗毅文,王斌,等.补肾活血汤治疗老年肱骨近端骨折围手术期的应用研究[J].中国中医骨伤科杂志,2016,24(10):16-19.
[17]GONG J,MENG HB,HUA J,et al.The SDF-1/CXCR4axis regulates migration of transplanted bone marrow mesenchymal stem cells towards the pancteas in rats with acute pancreatitis[J].Mol Med Rep,2014,9(5):1575-1582.
[18]GHADGE S K,MUHLSTEDT S,OZCELIK C,et al.SDF-1alpha as a therapeutic stem cell honing factor in myocardial infarction[J].Pharmacol Ther,2011,129(1):97-108.
[19]LISIGOLI G,TONEGUZZI S,GRASSI F,et al.Different chemokine are expressed in human arthritis bone biopsies:IFN-γand IL-6 differently modulate IL-8,MCP-1 and RANTES production by arghritis osteoblasts[J].Cytokine,2002,20(5):231-238.
[20]SIMTH H,WHITTALL C,WEKSLER B,et al.Chemokines stimulate bidirectional of human mesenchymal stem cells across bone marrow endothelial cells[J].Stem Cells Dev,2012,21(3):476-486.
[21]易诚青,马春辉,张国桥,等.CXCL13和CXCR5在骨缺损成骨微环境中的表达特征[J].上海交通大学学报:医学版,2012,32(12):1536-1539.
[22]李新建,齐振熙,李志敏,等.补肾活血汤含药血清骨髓间充质干细胞成骨-成脂分化的影响[J].中华中医药杂志,2016,24(8):3274-3277.
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